There have been known several carbohydrates which are composed of glucose molecules as constituents and produced from starches, amyloses, or partial starch hydrolyzates as amylaceous materials, for example, amylodextrins, maltodextrins, maltooligosaccharides, and isomaltooligosaccharides. These carbohydrates are also known to have usually both non-reducing and reducing groups at their molecular ends and the reducing group is responsible for reducibility. In general, reducing power of a partial starch hydrolyzate is represented with dextrose equivalent (DE), a scale for the reducing power, on a dry solid basis. Such a partial starch hydrolyzate with a high DE value has a low molecular weight, viscosity, strong sweetening power and reactivity: They easily react with amino group-containing substances such as amino acids and proteins through the amino carbonyl reaction which may lead to browning, undesirable smell, and deterioration. To overcome these disadvantages, heretofore long desired are methods which may lower or even eliminate the reducing power of partial starch hydrolyzates without converting glucose molecules as constituent saccharides. For example, it was reported in Journal of the American Chemical Society, Vol. 71, 353–358 (1949) that starches can be converted to α-, β- and γ-cyclodextrins which are composed of 6–8 glucose molecules linked covalently via the α-1,4 glucosidic linkage by allowing to contact with “macerans amylase”. Nowadays, cyclodextrins are produced on an industrial scale and their inherent properties such as non-reducibility, tasteless, and clathrating abilities render them very useful in a variety of fields. While, for example, Japanese Patent Kokai Nos. 143,876/95 and 213,283/95, filed by the same applicant of the present invention discloses a method of producing trehalose, a disaccharide composed of two glucose molecules linked together via the α,α-linkage, where a non-reducing saccharide-forming enzyme and a trehalose-releasing enzyme are allowed to contact with partial starch hydrolyzates such as maltooligosaccharides. In these days, trehalose has been industrially produced from starches and applied to a variety of fields where its non-reducibility, mild- and high quality-sweetness are advantageously utilized. As described above, trehalose (DP of two) and α-, β- and γ-cyclodextrins (DP of 6–8) have been produced on an industrial scale and extensively used because of their advantageous properties; however, there is a limitation in the types of non- or low-reducing saccharides which are available in the art. Therefore, saccharides other than these saccharides are in great demand.
Recently, reported was a novel cyclic tetrasaccharide composed of glucose units: European Journal of Biochemistry, Vol.226, 641–648 (1994) reported that a cyclic tetrasaccharide with the structure of cyclo{→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→} (which may be simply designated as “cyclotetrasaccharide”, hereinafter) is formed by allowing alternanase, a type of hydrolyzing enzyme, to contact with alternan, a polysaccharide where glucose molecules are linked via the alternating α-1,3 and α-1,6 bonds, followed by crystallization in the presence of methanol.
Cyclotetrasaccharide, a saccharide with a cyclic structure and no reducing power, is expected to be very useful because of its no amino-carbonyl reactivity, stabilizing effect for volatile organic compounds by its clathrating ability, and no apprehension of browning and deterioration.
However, alternan and alternanase, which are indispensable materials to produce cyclotetrasaccharide, are not easily obtainable, and microorganisms as alternanase source are not easily available.
Under these circumstances, the present inventors disclosed in WO 01/90338 Al a successful process to produce cyclotetrasaccharide where a saccharide with a glucose polymerization degree of 3 or higher and bearing both the α-1,6 glucosidic linkage as a linkage at the non-reducing end and the α-1,4 glucosidic linkage other than the linkage at the non-reducing end (may be called “α-isomaltosylglucosaccharide” throughout the specification) as a material is allowed to contact with an α-isomaltosyl-transferring enzyme which specifically hydrolyzes the linkage between the α-isomaltosyl moiety and the resting glucosaccharide moiety, and then the enzyme transfers the released α-isomaltosyl moiety to another α-isomaltosylglucosaccharide to form cyclotetrasaccharide. The α-isomaltosyl-transferring enzyme forms cyclotetrasaccharide from α-isomaltosylglucosaccharide by α-isomaltosyl-transferring reaction. Particularly, α-isomaltosyl-transferring enzyme has the following physicochemical properties:
(1) Action                Forming cyclotetrasaccharide with the structure of cyclo{→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→6)-α-D-glucopyranosyl-(1 3)-α-D-glucopyranosyl-(1→} from a saccharide with a glucose polymerization degree of 3 or higher and bearing both the α-1,6 glucosidic linkage as a linkage at the non-reducing end and the α-1,4 glucosidic linkage other than the linkage at the non-reducing end by catalyzing α-isomaltosyl-transferring reaction;        
(2) Molecular Weight                About 82,000 to 136,000 daltons when determined on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE);        
(3) Isoelectric Point (pI)                About 3.7 to 8.3 when determined on isoelectrophoresis using ampholine;        
(4) Optimum Temperature                About 45 to 50° C. when incubated at pH 6.0 for 30 minutes;        
(5) Optimum pH                About 5.5 to 6.5 when incubated at 35° C. for 30 minutes;        
(6) Thermal Stability                About 45° C. or lower when incubated at pH 6.0 for 60 minutes; and        
(7) pH Stability                About 3.6 to 10.0 when incubated at 4° C. for 24 hours.        
As regards to saccharides which are used as starting materials for cyclotetrasaccharide, it is desirable to prepare them from starches which are abundant and low-cost sources, however, since α-isomaltosyl-transferring enzyme dose not directly act on starches, the following procedures are actually employed: Starches are firstly converted into an α-isomaltosylglucosaccharide having the above specified structure, for example, relatively low-molecular weight isomaltooligosaccharide such as panose and isomaltosylmaltose, and then subjected to the action of α-isomaltosyl-transferring enzyme to form cyclotetrasaccharide. As regards to the yield of cyclotetrasaccharide from the materials, in the case of using panose as a material, the yield of cyclotetrasaccharide is about 44% based on the weight of the dry solid (d.s.b.). Similarly, in the case of using isomaltosylmaltose as a material, the yield of cyclotetrasaccharide is about 31%, d.s.b. While in the case of using starches as a material, it is necessary to contact starches previously with α-amylase, starch-debranching enzyme, β-amylase and α-glucosidase to form relatively low-molecular weight isomaltooligosaccharides including panose, and the yield of cyclotetrasaccharide is relatively low, about 15%, d.s.b. Although the production of cyclotetrasaccharide from starch is feasible even in such a low yield, the production cost may be increased. Therefore, it is desired to establish a novel method for producing cyclotetrasaccharide in a relatively high yield using starches as a material.
Under these circumstances, the present inventors extensively screened microorganisms capable of producing an α-isomaltosylglucosaccharide-forming enzyme which may significantly improve the yield of cyclotetrasaccharide when allowed to act on starches as a material. As a result, the present inventors found that α-isomaltosyl-transferring enzyme-producing microorganisms, strains C9, C11, N75 and A19 of the genera Bacillus and Arthrobacter, which are disclosed in WO 01/90338 A1, also produce another α-isomaltosylglucosaccharide-forming enzyme. They also found that the yield of cyclotetrasaccharide can be remarkably improved by allowing both α-isomaltosylglucosaccharide-forming enzyme and α-isomaltosyl-transferring enzyme to act on a glucosaccharide with a high-molecular weight such as partial starch hydrolyzates. The present inventors characterized the α-isomaltosylglucosaccharide-forming enzyme, and established a process for producing the enzyme. Further, they established methods for α-glucosyl-transferring reaction using the enzyme, a process for producing α-isomaltosylglucosaccharide, and a process for producing cyclotetrasaccharide and a saccharide composition containing the cyclotetrasaccharide by the combination use of the enzyme and α-isomaltosyl-transferring enzyme. Also, the present inventors established food products, cosmetics and pharmaceuticals, comprising cyclotetrasaccharide which are obtainable by the processes mentioned above or saccharide compositions containing cyclotetrasaccharide. However, since the producibility of α-isomaltosylglucosaccharide-forming enzyme in the microorganisms were found to be not enough, there has been still left a problem that large-scale cultivation of such microorganisms as enzyme sources are required for industrial scale production of α-isomaltosylglucosaccharide and cyclotetrasaccharide.
It is known that the entity of the enzyme is a polypeptide and the enzymatic activity is under the regulation of its amino acid sequence, which is encoded by a DNA. Therefore, if one successfully isolates a gene which encodes the polypeptide and determines the nucleotide sequence, it will be relatively easy to obtain the desired amount of the polypeptide by the steps of constructing a recombinant DNA which contains the DNA encoding the polypeptide, introducing the recombinant DNA into host-cells such as microorganisms, animals or plants, and culturing the obtained transformants in appropriate nutrient media. Under these, required are the isolation of a gene encoding the polypeptide as the entity of the enzyme described above, and the sequencing of the nucleotide sequence.